The HOX homeodomain (HD) proteins are key regulators of hematopoiesis. Aberrant expression or chromosomal translocations involving certain HOX proteins, such as HOXA9, have been implicated in the pathogenesis of acute myeloid leukemia (AML). Our understanding of how the intracellular levels and activities of the HOX proteins are controlled during hematopoiesis is current limited to transcriptional regulation and signal transduction. Little isknown about the posttranslational control mechanisms that govern the abundance of the HOX hematopoietic regulators and the functional significance for such regulations. The long-term goal of this study is to understand how ubiquitin- dependent protein degradationregulates normal and malignant hematopoiesis. The central hypothesis of the application is that the cullin 4A (CUL-4A) ubiquitin-ligase controls hematopoietic development throughtargeted degradation of key hematopoietic regulators. The hypothesis has been formulated on the basis of strong preliminary data, which demonstratedthat CUL-4A targets HOXA9 for degradation, and regulates myeloid differentiationand maturation. Hematopoietic-specific knockout of the CUL-4A gene in mice led to an increased expansion of bone marrow progenitor cells and peripheral blood leukocytes. This proposal seeks to determine the biochemical mechanisms underlying the CUL-4A-dependent proteolytic control of HOXA9 and the chromosomal translocation- derived NUP98-HOXA9 fusion, and to elucidate the functional significance of CUL-4A in suppressing leukemic transformation. We are uniquely prepared to undertake the proposed research, since we have recently generateda CUL-4A-resistant HOXA9 mutant, and developed conditional CUL-4A knockout mice and CUL-4A siRNA to eliminate or modulate CUL-4A activity. We have also optimized lentiviral- and retroviral-based gene delivery systemsfor efficient transduction in primary hematopoietic stem and progenitor cells. We propose to combine the biochemical and molecular genetic approachesin Dr. Pengbo Zhou's lab and the expertise in ex vivo and in vivo hematopoieticanalysis in Dr. Malcolm Moore's lab to address the following specific aims: (1) to define the biochemical mechanisms underlying CUL~4A-dependent ubiquitination and degradation of HOXA9. (2) to elucidate the functional significance of HOXA9 degradation by CUL-4A in the pathogenesis of AML. (3) to determine the molecular basis for CUL-4A resistanceby the leukemogenic NUP98-HOXA9fusion and to assess the impact of CUL-4A ablation in the mouse model of NUP98- HOXA9-induced leukemia. Since little information is available regarding the roles of protein degradation during leukemogenesis, successful completion of this proposal will represent a significant advance in understanding a novel posttranslational mechanism that governs the functions of key hematopoietic regulators, and provide a frameworkfor future investigations of targeted protein degradation in normal and malignant hematopoiesis.